Granulocyte colony-stimulating factor (G-CSF or GCSF) is a glycoprotein that stimulates the bone marrow to produce granulocytes and release them into the bloodstream. In biotherapeutics, G-CSF showed efficacy in treatment of neonatal infections, granulocyte transfusion in patients with neutropenia, in severe infections and sepsis, in acute myeloid leukaemia's made it an essential biopharmaceutical drug. Commercially, two forms of recombinant human G-CSF are available that include Escherichia coli (E. coli)-derived G-CSF, which has no sugar chain (non-glycosylated G-CSF; filgrastim; Neupogen, Amgen) and Chinese hamster ovary cell derived-CSF (glycosylated G-CSF; lenograstim, Chugai Pharma UK Ltd).
Filgrastim is a water-soluble 175 amino acid protein with a molecular weight of 18,800 Daltons. Filgrastim is obtained from the bacterial fermentation of a strain of Escherichia coli (E. coli) transformed with a genetically engineered plasmid containing the human G-CSF gene. The biological activities of G-CSF includes stimulation and differentiation of progenitor “stem cells” into a variety of blood cell lines, stimulation of the proliferation of differentiated blood cell lines and stimulating the ultimate differentiation of mature blood cells from proliferated cell lines.
The production of recombinant therapeutic proteins using microorganisms as host system is often difficult because the high-level expression of recombinant proteins leads to the formation of inclusion bodies (IBs) that are insoluble aggregates, as well as the recombinant proteins are present in biologically inactive form which require additional downstream steps to make the protein biologically active and suitable for further purification steps. Moreover, it is often difficult to recover the protein from IBs because of the issues concerned with the initial recovery, solubilization and renaturation steps. The production of recombinant proteins from inclusion bodies can be viable if a simple and cost-effective downstream process may be developed.
The expanding demand for biopharmaceuticals has catalyzed advancement in both upstream as well as downstream processing of biotherapeutic proteins. Significant improvements have been made in the cell culture titers in the last few years and this has moved the centre of bio-pharmaceutical development towards improving the commercial concerns of downstream processing. Protein biotherapeutics are typically produced alongside an assortment of impurities. These include host cell related impurities, process impurities and product related impurities/variants. Of these, the product related impurities/variants are difficult to remove as their physicochemical properties are very similar to the product itself however as these impurities/variants can significantly affect the biological activity of the target therapeutics.
The above mentioned variants/impurities include different oxidation forms of methionine (Met) residues of G-CSF. It is known that, G-CSF protein contains four methionine residues at Met1, Met122, Met127 and Met138 positions. It is observed that at different oxidative conditions, each of the four methionine residues oxidizes at different rates [Met1>Met138>Met127>Met122] which may be resolved by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) chromatogram to native G-CSF. In addition, impurities like reduced and aggregated form of G-CSF formed by misfolding of native G-CSF demonstrate additional peaks in RP-HPLC chromatogram. These and other such forms of undesired G-CSF leads to reduction in biological activity of native G-CSF. Therefore it is crucial in the pharmaceutical development of therapeutic proteins to remove such impurities during purification process. Furthermore, in RP-HPLC chromatogram, an extra peak of N-formyl methionine variant of G-CSF is also observed along with native G-CSF that possesses the same biological activity as that of native form but sometimes may lead to immunogenic response in patient. The N-formyl methionine variant is a result of partial retention of the formyl group by def gene of E. coli due to high level expression of the recombinant proteins. This variant is also an impurity and need to be removed. Thus, its effective removal is a key objective for the purification platforms for bacterial based production of therapeutic proteins.
For removing the aforementioned product related impurities, various chromatographic techniques were used in prior art such as multimodal chromatography which involves the use of resins that offer a combination of interactions between the product and resin like Hydroxy apatite (HA) (cation exchange and metal affinity interaction), Capto MMC (cation exchange and hydrophobic interactions), Capto Adhere (anion exchange and hydrophobic interactions), and HEA/PPA. All of these resins are effective and provide different selectivities but are costly when compared to conventional ion exchange and hydrophobic resins, thereby increasing the cost of the final product. Moreover, optimization of process parameters with multimode resins is a tedious and time consuming process which increases the process development time and subsequently the running time for chromatography.
In last few years development and manufacturing of therapeutic G-CSF involves numerous purification schemes. For instance, WO1987/03689 discloses the use of immune-affinity chromatography for isolation and purification of recombinant G-CSF which was not a well accepted approach for commercial manufacturing, since it could raise its own regulatory concerns. Also the cost of immune-affinity chromatography media was very high compared to conventional chromatography matrices owing to use of monoclonal antibodies for their preparation.
European patent EP2341061, discloses a purification process involving a series of four chromatography steps, comprising two gel filtration chromatographies, a cation exchange chromatography and an anion exchange chromatography for preparation of G-CSF.
Another European patent EP1904522, discloses a purification process used for purification of G-CSF primarily involving three chromatography steps namely, two cation exchange chromatography and a hydrophobic interaction chromatography.
WO 2001/04154 discloses a purification process which includes a hydrophobic interaction chromatography, a hydroxyl apatite chromatography and a cation exchange chromatography to purify the G-CSF. For E. coli-derived G-CSF, solubilization of inclusion bodies and refolding of G-CSF are extra steps to be considered. On a commercial scale, due to the multistep process, the final yield decreases to a great extent. Hence a simplified process with fewer steps with higher yields in a shorter time is required.
In prior art, the purification processes described are complicated, long and includes multiple chromatography steps to get the purified G-CSF. Furthermore, none of the earlier processes reveal a basic, prudent and financially practical strategy for the production of G-CSF which could ensure the consistent production of stable product at industrial scale. Subsequently, to conquer the significant issues concerned with the production of G-CSF, a simple, scalable, commercially viable and steadier process for high recovery of G-CSF has been currently developed and disclosed in the present invention. The process of the present invention comprises, streamlined, orthogonal, robust and scalable downstream process steps for production of G-CSF at industrial scale with higher yield and purity.